In recent years, using micro-machine technology and microscopic processing technology, systems are developed in which devices and means, for example, pumps, valves, flow paths, sensors and the like for performing conventional sample preparation, chemical analysis, chemical synthesis and the like are miniaturized and integrated on a single chip.
These systems are called μ-TAS (Micro Total Analysis System), bioreactor, lab-on-chips, and biochips, and much is expected of their application in the fields of medical testing and diagnosis, environmental measurement and agricultural manufacturing.
As seen in genetic screening in particular, in the case where complicated steps, skilful operations, and machinery operations are necessary, a microanalysis system, which is automatic, has high speed and is simple, is very beneficial not only in terms of reduction in cost, required amount of sample and required time, but also in terms of the fact that it makes analysis possible in cases where time and place cannot be selected.
At a site where various testing such as clinical testing is carried out, even in a case of measuring with a microreactor of a chip type which can quickly output results regardless of place, quantitation and accuracy in analysis are deemed to be important.
In various analysis and tests, quantitation of analysis, precision of analysis and economy are major factors in the development of the aforementioned analysis chip capable of producing results independently of place. To achieve this purpose, it is important to establish a highly reliable liquid feed system of simple structure. Thus, there has been an active demand for a reliable, high-precision micro fluid control device. The present inventors have already proposed a micro pump system and a control method capable of meeting such requirements (Patent Documents 2 to 4).
In order to cause reaction between a specimen and reagent and transport a reaction product to a detection part by the use of such a micro pump and detect the reaction product such that the related operations can be performed in a single chip, a serial micro flow path is formed in the chip. In this case, it is necessary to integrate and dispose a flow path system including storage sections for a specimen and reagents, a mixing section to mix the reagents, a reaction section, a detection section, and flow paths to communicate these sections, and accompanying function elements, densely in a limited space.
Furthermore, the inventors of the present invention have already proposed, in Patent Document 5 (Japanese Patent Application TOKUGAN No. 2004-138959), a testing microchip (microreactor) including: a specimen storage section in which specimen is stored; a reagent storage in which reagent is stored; a reaction section which has a reaction flow path in which the specimen stored in the specimen storage section and the reagent stored in the reagent storage section are merged to perform a predetermined reaction processing; and a testing section which has a testing path for performing a predetermined test on the reaction-processed substance obtained from the reaction in the reaction section, wherein the specimen storage section, the reagent storage section, the reaction section, and the testing section are connected continuously by a serial flow path from the upstream side to the downstream side.
That is, a testing micro chip 100 of a prior art, as disclosed in Patent Document 5, includes a specimen storage section 104 for storing a specimen 102 and reagent storage sections 108 in which reagents 106 are sealed in advance.
The micro chip 100 also includes a mixing flow path 110 in which the reagents 106 stored in the reagent storage sections 108 are mixed. The mixed reagent mixed in the mixing flow path 110 and the specimen 102 from the specimen storage section 104 are mixed through a Y shape flow path or the like. A reaction flow path 112 is provided in which reaction is started by a heater 116 or the like.
Further, the specimen having been caused to react with the mixed reagent in the reaction flow path 112 is transported to the downstream side of the flow path 114 and the reaction is detected at a detection part that is provided on an analysis flow path 114.
With such a testing micro chip of a prior art, in a case, foe example, where a testing micro chip 100 performs an amplification reaction by ICAN method (Isothermal chimera primer initiated nucleic acid amplification), the specimen storage section 104 stores a specimen extracted from blood or sputum.
On the other hand, biotin-modified chimera-primer, DNA polymerase having a strand displacement activity and a reagent containing endonuclease hybridize are stored in the reagent storage sections 108, to specifically hybridize with a gene being a detection object.
Accordingly, when the mixed reagent having been mixed in the mixing flow path 110 and the specimen 102 from the specimen storage section 104 are mixed through a Y shape flow path or the like and mixed in the reaction flow path 102, gene amplification reaction is promoted in such a manner that a heater 116 which is, for example, a nichrome wire heater, sheathed heater, heater formed with an ITO membrane or a metallic membrane (chrome, gold, platinum, etc.) on a substrate is applied under heat control to the range 50 to 65° C., for example, to 55° C.
With an example of an amplification reaction by a PCR (polymerase chain reaction) method, it is necessary to heat a flow path that constructs the reaction part and also, if necessary, a flow path which performs preprocessing, to a predetermined temperature. On the other hand, there are also regions which are desirably not to rise in temperature, in the serial micro flow path including a number of function parts. For example, many of specimens and reagents tend to denature when heated and need cooling to avoid it. Parts where such specimens or reagents are stored or parts where reagents are mixed with each other require selective heat dissipation or cooling.
In a chip that contains an integrated serial micro flow path in a limited space, in a case where a flow path which is adjacent to a reaction part to be heated and for which a rise in temperature is undesirable is disposed, or in a case where non-satisfactory temperature distribution may cause a problem on reaction, it is necessary to prevent transfer of heat from the heated part to flow paths near the section. For example, when a part of a liquid in a flow path is heated and if meniscus (the front end boundary surface of a liquid in a flow path) of a liquid is present in a heated area, to be heated, or its vicinity, liquid may evaporate from the meniscus. This may affect the accuracy of quantitation and cause various problems. Especially, in a case where liquid is fed to a successive reaction process section after a heating process of the liquid in a μTAS is completed at a heating section, it is necessary to keep the liquid at the place where it is in the heated area for a certain time, which tends to cause the above described problems.
[Patent Document 1]TOKKAI No. 2004-28589[Patent Document 2]TOKKAI No. 2001-322099[Patent Document 3]TOKKAI No. 2004-108285[Patent Document 4]TOKKAI No. 2004-270537[Patent Document 5]TOKUGAN No. 2004-138959    [Non-patent Document] “DNA Chippu-gijutu To Sono Ouyou (DNA chip technology and application)” and No. 13 of Volume 43 of “Tanpakushitu Kakusan Kouso (Protein, Nucleic acid and Enzyme)” (1998) written by Fusao Kimizuka and Ikunoshin Kato and published by Kyoritsu Shuppan Co., LTD.
However, heating with these kinds of heaters may cause gas bubbles in the mixed liquid between a specimen and reagent if the heating time by the heater is long. The gas bubbles function to inhibit coupling between biotin-modified chimera-primer that specifically hybridizes with a gene being a detection object and a specimen, which may prohibits a predetermined test in a testing section.
Further, long time heating with these kinds of heaters causes a side-reaction other than reaction between a specimen and reagent. That is, a side-reaction by various materials other than a target material occurs, and amplified products of which inhibit amplification of the target material. Consequently, analysis based on reaction as a predetermined purpose becomes difficult and a predetermined test in a testing section may not be achieved.
Still further with these kinds of heaters, even if heating is stopped, a certain time is required before a heater is cooled down. With such a residual heat time, gas bubbles and side reaction, as described above, may be caused by the inertia temperature.
Yet further, since the reagent 106 stored in the reagent storage section 108 has a characteristic of denaturing due to effect by temperature, such a long heating time and residual heat time may denature the reagent 106 stored in the reagent storage section 108, making it impossible to carry out a predetermined test in the testing section.
The present invention wad devised, taking problems into account, such as described above. An object of the invention is to provide a micro integrated analysis system and a method by the system which inhibit transfer of residual heat from a heating part to neighboring flow paths in a testing chip as much as possible, and includes a flow path structure that makes the temperature distribution in the chip to be rigorous as much as possible.
Further, another object of the invention is to provide a testing micro chip and a micro integrated analysis system using the chip, which are reliable. In this micro chip, when a mixed reagent having been mixed in a mixing path and a specimen from a specimen storage section meet with each other and get mixed in a mixing and reaction flow path, it is possible to quickly heat the mixture to a necessary temperature, to heat the mixture for a necessary time required for reaction when the temperature has reached the necessary temperature, and to quickly cool the mixture so that no gas bubbles are generated and no side reaction is caused. Thus, an accurate and correct test can be achieved.